Fig 2: HAT1 knockdown re-sensitizes CRPC cells to enzalutamide treatment. (A and B) 22Rv1 cells were infected with lentivirus vectors expressing control or mixed HAT1-specific shRNAs for 48 h and selected with puromycin (10 µg/ml) for another 72 h. The cells were then harvested for western blot analysis (A) and qRT-PCR analysis (B). (C) The schematic diagram demonstrating the drug treatment method for mice bearing subcutaneous tumors. (D-G) 22Rv1 cells from (A) were mixed with Matrigel and injected subcutaneously (s.c.) into the right dorsal flanks of NOD/SCID mice and then treated with enzalutamide (10 mg/kg, p.o.) every 2 days when the average size of tumors reached 50 mm3. The growth curves of tumors were measured (D), and the tumors were harvested at day 27 and photographed (E). The mice (F) and tumors (G) were weight. (H) H&E staining and IHC staining for HAT1, Ki-67 and cleaved caspase 3 were performed. Representative images were taken from each group. Scale bars are indicated in the images, scale bar = 50 µm. (I and J) Ki-67 (I) and cleaved caspase 3 (J) positive cells in tissue sections obtained from (H) were quantified. The number of positive cells from at least five fields was counted and analyzed (I and J). Data were presented as the mean ± SD of three independent experiments (A-G). Error bars indicate SD. The p values were analyzed using Student's t-test. **p < 0.01, ***p < 0.001

Fig 3: HAT1 regulates AR expression transcriptionally in PCa cells. (A) LNCaP, C4-2, and 22Rv1 cells were infected with lentivirus vectors expressing control or HAT1-specific shRNAs for 48 h and selected with puromycin (10 µg/ml) for another 72 h. The cells were then harvested for western blot analysis. (B-E) LNCaP, C4-2, and 22Rv1 cells were infected with lentivirus vectors expressing control or HAT1-specific shRNAs for 48 h and selected with puromycin (10 µg/ml) for another 72 h. The cells were then harvested for qRT-PCR analysis. (F and G) LNCaP and 22Rv1 cells were transfected with the indicated volume of plasmids, then cells were harvested for western blot analysis (F) and qRT-PCR analysis (G) 24 h post-transfection. (H and I) C4-2 and 22Rv1 cells were treated with the indicated concentrations of ascorbate for 24 h, and cells were harvested for western blot analysis (H) and qRT-PCR analysis (I). (J and K) LNCaP and 22Rv1 cells were infected with lentivirus vectors expressing control or HAT1-specific shRNAs for 48 h, selected with puromycin (10 µg/ml) for another 72 h, and then treated with or without ascorbate for 24 h. Cells were harvested for western blot analysis (J) and qRT-PCR analysis (K). Data were presented as the mean ± SD of three independent experiments. Error bars indicate SD. The p values were analyzed using ANOVA. ***p < 0.001

Fig 4: HAT1 increases AR expression through BRD4 in PCa cells. (A and B) 22Rv1 cells were infected with lentivirus vectors expressing control or HAT1-specific shRNAs for 48 h and selected with puromycin (10 µg/ml) for another 72 h. The cells were then treated with or without JQ1 (3 µM) for 24 h and harvested for western blot analysis (A) and qRT-PCR analysis (B). (C) The condition of binding BRD4 to the AR promoter in UCSC Genome-Browser ChIP-sequence data. Red arrow denotes the binding peak. (D) Correlation of BRD4 and AR mRNA expression in human PCa samples was determined by the GEPIA webtool. (E and F) 22Rv1 cells were infected with lentivirus vectors expressing control or HAT1-specific shRNAs or BRD4-specific shRNAs for 48 h and selected with puromycin (10 µg/ml) for another 72 h. The cells were then harvested for western blot analysis (E) and qRT-PCR analysis (F). (G and H) 22Rv1 cells were infected with lentivirus vectors expressing control or BRD4-specific shRNAs for 48 h and selected with puromycin (10 µg/ml) for another 72 h. The cells were then transfected with the indicated plasmids for 48 h. Cells were harvested for western blot analysis (G) and qRT-PCR analysis (H). (I) 22Rv1 cells were infected with lentivirus vectors expressing control or HAT1-specific shRNAs for 48 h and selected with puromycin (10 µg/ml) for another 72 h. The cells were then treated with or without JQ1 (3 µM) for 24 h and harvested for ChIP-qPCR analysis. (J) A hypothetical model depicting the mechanism of HAT1 catalyzing histone H4 acetylation and recruiting BRD4 binding to the acetylated H4 and initiating AR transcription. Data were presented as the mean ± SD of three independent experiments. Error bars indicate SD. The p values were calculated using ANOVA (B and I) and Student's t-tests (F and H). Spearman's correlation analysis was performed to determine the correlation between AR and BRD4, and the p value is shown as indicated (D). **p < 0.01, ***p < 0.001

Fig 5: AR expression in patient specimens is positively correlated with HAT1. (A) Images for IHC staining of HAT1 and AR in PCa tissue sections. Scale bars are shown as indicated. (B and C) The correlation analysis (B) (n = 31) and heatmap analysis (C) of HAT1 and AR from the IHC staining scores in (A) are shown. (D) Correlation of HAT1 and AR mRNA expression in human PCa samples was determined by the GEPIA webtool. The co-efficiency r or R and p values were calculated using Pearson's correlation (B) and Spearman's correlation (D) analyses, respectively and are shown as indicated